Human immunodeficiency virus type 1 (HIV-1) has been infecting humans for ~20 years now with little hope for an efficacious vaccine in the near future (Ho 2002). Alternative strategies to inhibit viral replication either alone or in combination with the currently administered therapies are tremendously desirable. One such strategy involves the use of small interfering 21-26 bp double stranded RNAs (siRNAs) which have recently been shown to be ubiquitous and specific repressers of gene expression (reviewed (Finnegan and Matzke 2003)). Of particular interest is the use of siRNAs to specifically target genes involved in diseases such as HIV-1. Importantly, the use of these siRNAs to target HIV-1 or its chemokine co-receptor at the level of promoter-controlled expression (i.e. transcriptional gene silencing, TGS) would be an immeasurable and potent therapeutic agent. Until recently there was no published data suggesting that TGS is operable in mammalian cells and the majority of siRNA activity is cytoplasmic (Zeng 2002). Recently we demonstrated in human cells that siRNAs constructed to target a promoter can effectively suppress a genes mRNA expression (Morris, Chan et al. 2004). Moreover, the preliminary data presented here adds further support to the observation that siRNAs can induce TGS in human cells only upon successful delivery of the siRNA to the nucleus and that the pathway of the observed inhibition involves histone deacetylation (HDAC). We hypothesize that the HIV-1 promoter, specifically the 5'LTR, and the promoter for the chemokine co-receptor CCR5 used for entry by HIV-1 can be specifically targeted by siRNAs (delivered directly into the nucleus) and that these promoters undergo epigenetic changes that are specific, long lasting, and significantly alter the transcriptional profile of the integrated virus or chemokine co-receptor. To test this hypothesis we propose to 1) determine the magnitude, duration, and specificity of siRNA mediated suppression by targeting a minimum of 4 sites in the HIV-1 LTR and CCR5 promoter, 2) Characterize promoter specific siRNA mediated suppression of HIV-1 viral replication (LTR specific siRNAs) or HIV-1 entry (CCR5 specific siRNAs) in transduced cells challenged with HIV-1, 3) characterize the epigenetic changes (DNA methylation, histone acetylation/ phosphorylation and methylation) associated with siRNA mediated TGS of the HIV-1 LTR and CCR5 promoter, and 4) determine the mechanism of action of the siRNA mediated TGS by knocking out DNMT-1 and HDAC's as well as determine if an interaction with Sin3 or Mi2/NuRD protein complexes is involved. The work outlined in this project should further develop our understanding of this complex molecular pathway of target specific gene regulation in human cells, specifically HIV-1 and it's chemokine co-receptor CCR5, and provide findings that will be applicable in future therapeutic interventions aimed at altering patterns of HIV-1 and co-receptor expression.